Biocompatible magnetic nanosensors have been designed to detect molecular interactions in biological media. Upon target binding, magnetic nanosensors cause changes in spin-spin relaxation times of neighboring water molecules (or any solvent molecule with free hydrogens) of a sample, which can be detected by classical magnetic resonance (NMR/MRI) techniques. By using nanosensors in a sample, it is possible to detect the presence of an analyte at very low concentration—for example, small molecules, specific DNA, RNA, proteins, carbohydrates, lipids, lipoproteins, organisms, and pathogens (e.g. bacteria, viruses, etc.)—with sensitivity in the low femtomole range (e.g., about 0.5 fmol to about 30 fmol per microliter; less than ten analyte particles (e.g., virus/cell) per microliter).
In general, magnetic nanosensors used are superparamagnetic nanoparticles functionalized with affinity moieties that bind or otherwise link to their intended molecular target to form clusters (aggregates) or nanoassemblies. It is thought that when superparamagnetic nanoparticles assemble into clusters and the effective cross sectional area becomes larger, the nanoassembly becomes more efficient at dephasing spins of surrounding water (or other solvent) protons, leading to an enhancement of measured relaxation rates (1/T2). Additionally, nanoassembly formation can be designed to be reversible (e.g., by temperature shift, chemical cleavage, pH shift, etc.) so that “forward” or “reverse” assays can be developed for detection of specific analytes. Forward (clustering) and reverse (declustering) types of assays can be used to detect a wide variety of biologically relevant materials. Furthermore, spin-lattice relaxation time (T1) is considered independent of nanoparticle assembly formation and can be used to measure concentration in both nano-assembled and dispersed states within the same solution.
Examples of magnetic nanosensors are described in Perez et al., “Use of Magnetic Nanoparticles as Nanosensors to Probe for Molecular Interactions,” ChemBioChem, 2004, 5, 261-264, and in U.S. Patent Application Publication No. US2003/0092029 (Josephson et al.), the texts of which are incorporated by reference herein, in their entirety. Examples of magnetic nanosensors include monocrystalline iron oxide nanoparticles from about 3 to about 5 nm in diameter surrounded with a dextran coating approximately 10 nm thick such that the average resulting particle size is from about 25 to about 100 nm. Another example of magnetic nanosensors include polycrystalline iron oxide nanoparticles of about 100 nm to about 1 micron in diameter.
Nanosensors have demonstrated low femtomolar analyte detection sensitivity through cluster formation (i.e. aggregation) and dispersion (i.e. disaggregation) assays. However, sensivity is just one requirement for a versatile bioanalytical detection system. A versatile bioanalytical detection system should also provide rapid results and be adaptable to functioning with a wide range of analyte concentrations for a variety of bioanalytical assays. Aggregation/disaggregation of nanosensors may not be the optimal method for analyte detection in all assays.
For example, cluster formation can only occur when each nanoparticle is bound to multiple analytes and, in some cases, each analyte is bound to multiple nanoparticles. Additionally, aggregation can be inhibited by geometrical effects such as a variation in size among nanosensors and analytes. Further, long incubation time may be required for cluster formation due to a two-step kinetic process for aggregation. Analyte needs to first bind to one or more nanosensor(s), then nanosensors agglomerate with each other to form clusters.
Cluster formation has also been shown to limit the dynamic range for certain bioanalytical assays. Factors that may contribute to limiting dynamic range include the structural instability of clustered aggregates. In addition, excess aggregation may lead to precipitation of nanosensors out of solution. Further, imperfect magnetization coupling of nanoparticles with each other, over an extended period of incubation time, may also contribute to a reduction in net magnetization per unit volume of a cluster, making relaxation process less efficient and lowering its magnitude. Therefore, the need exists for designs of versatile nanosensors and bioanalytical assays that exploit the individual magnetic nanoparticle's enhanced capability of dephasing spins of water protons for analyte detection without aggregation.